U.S. patent application number 12/410040 was filed with the patent office on 2009-09-03 for rapid efficacy assessment method for lung cancer therapy.
This patent application is currently assigned to National Health Research Institutes. Invention is credited to Konan Peck, Yuh-Pyng Sher, Jin-Yuan Shih, Cheng-Wen Wu, Pan-Chyr Yang.
Application Number | 20090220985 12/410040 |
Document ID | / |
Family ID | 37804685 |
Filed Date | 2009-09-03 |
United States Patent
Application |
20090220985 |
Kind Code |
A1 |
Peck; Konan ; et
al. |
September 3, 2009 |
RAPID EFFICACY ASSESSMENT METHOD FOR LUNG CANCER THERAPY
Abstract
The present invention discloses a method for rapid assessment of
lung cancer therapy efficacy in a few days instead of weeks by
conventional imaging methods. This method can also be used to
detect relapse of the cancer and to improve the current TNM cancer
staging method for more accurate prognosis. The rapid assessment of
therapy efficacy is based on detecting circulating cancer cells in
body fluid with high positive detection rate. The high positive
detection rate is achieved by using qPCR amplification of multiple
marker genes identified by in silico search of DNA sequence
database. This invention also discloses a scoring method to
calculate the cancer cell load based on qPCR results to correlate
the amount of circulating cancer cells in lung cancer patients and
predict the treatment outcomes.
Inventors: |
Peck; Konan; (Taipei City,
TW) ; Sher; Yuh-Pyng; (Taipei City, TW) ;
Shih; Jin-Yuan; (Taipei City, TW) ; Yang;
Pan-Chyr; (Taipei City, TW) ; Wu; Cheng-Wen;
(Zhunan Town, TW) |
Correspondence
Address: |
OCCHIUTI ROHLICEK & TSAO, LLP
10 FAWCETT STREET
CAMBRIDGE
MA
02138
US
|
Assignee: |
National Health Research
Institutes
Zhunan Town
TW
|
Family ID: |
37804685 |
Appl. No.: |
12/410040 |
Filed: |
March 24, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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|
11306532 |
Dec 31, 2005 |
7507534 |
|
|
12410040 |
|
|
|
|
60596104 |
Sep 1, 2005 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 2600/118 20130101;
C12Q 1/6886 20130101; C12Q 2600/112 20130101; C12Q 2600/106
20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for cancer relapse detection which comprises: (a)
collecting a blood or pleural effusion sample from a human subject;
(b) extracting total RNA from said sample; (c) amplifying a panel
of gene transcripts of said total RNA by qPCR, wherein said panel
of gene transcripts, named j, comprises keratin 19 (KRT19),
ubiquitin thiolesterase (UCHL1), tripartite motif-containing 28
(TRIM28), and highly similar to HSFIB1 for fibronectin; (d)
measuring a qPCR threshold cycle number (C.sub.T).sub.j for each
gene transcript of j in said panel of gene transcripts of said
total RNA from said sample and a qPCR threshold cycle number
C.sub.T.sup.(GAPDH) for the control gene transcript of said total
RNA from said sample; (e) calculating a differential expression
ratio Q.sub.j for each gene transcript of j in said panel of gene
transcripts of said total RNA according to
Q.sub.j=2.sup.(.DELTA.C.sup.T.sup.).sup.j.sup.-(.DELTA.C.sup.T.sup.).sup.-
j.sup.,mean wherein
(.DELTA.C.sub.T).sub.j=C.sub.T.sup.(GAPDH)-(C.sub.T).sub.j, and
(.DELTA.C.sub.T).sub.j, mean is a predetermined mean of
(.DELTA.C.sub.T).sub.j for gene transcript of j over a population
of persons not inflicted with lung cancer; (f) calculating a
normalized expression ratio E.sub.j for each gene transcript of j
in said panel of gene transcripts of said total RNA according to
E.sub.j=(Q.sub.j-Q.sub.j,mean)/.sigma..sub.j wherein Q.sub.j, mean
is a predetermined mean of differential expression ratio for gene
transcript of j over a population of lung cancer patients and
.sigma..sub.j is a predetermined standard deviation of differential
expression ratio for gene transcript of j over said population of
lung cancer patients; (g) calculating a load of cancer cells (Lc)
according to Lc=.SIGMA.E.sub.j, where the summation is over all the
gene transcripts in said panel of gene transcripts; (h) comparing
said Lc from step (g) with a reference load of cancer cells to
detect if cancer relapse has occurred in said human subject,
wherein said Lc from step (g) higher than said reference load of
cancer cells indicates that circulating cancer cells are present in
said human subject.
2. A reagent kit for assessment of lung cancer therapy and relapse
detection, comprising a panel of PCR primers and probes for the
multiple gene transcripts used in real-time quantitative PCR and a
scoring method.
3. A reagent kit of claim 2, wherein said multiple gene transcripts
are nucleic acid sequences.
4. A reagent kit of claim 2, wherein said nucleic acid sequences
are selected from Homo sapiens keratin 19 (KRT19), ubiquitin
thiolesterase, tripartite motif-containing 28 (TRIM28) and gene
highly similar to HSFIB1 for fibronectin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Under 37 CFR .sctn. 120, this application is a division of
U.S. application Ser. No. 11/306,532 filed Dec. 31, 2005, which
claims the benefit of U.S. Provisional Application No. 60/596,104,
filed Sep. 1, 2005, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to methods for
assessment of carcinoma cancer therapy and relapse detection, and
more specifically it relates to an efficacy assessment method for
lung cancer therapy to rapidly predict the outcome of lung cancer
therapy so treatment with higher likelihood of success can be
selected to prevent invalid treatment from wrecking patents, and a
routine monitoring method for cancer relapse after the
treatment.
[0004] 2. Description of the Related Art
[0005] Lung cancer is the leading cause of cancer-related death and
non-small cell lung cancer (NSCLC) accounts for .about.80% of the
cases. Attempts to use serum protein markers for the early
diagnosis of lung cancer have not yielded satisfactory results for
routine screening, and newly developed early diagnostic methods
using serum DNA as a diagnostic marker await further validation.
Current therapeutic measures remain unable to lower the mortality
rate of late-stage lung cancer patients. Surgical resection is
still the best cure for the early-stage patients. The tumor, node,
metastasis (TNM) classification has been used for cancer staging
and prognosis for decades. A large portion of early-stage patients,
defined by the current staging system and available imaging
modalities, still develop distant metastases although they received
surgical removal of the tumor mass. The inability to detect
disseminated tumor cells with the current imaging techniques is a
major obstacle to accurate cancer staging.
[0006] NSCLC is heterogeneous with respect to histology and
biological characteristics. Individual NSCLC cells within a tumor
and in different patients' tumors express different amounts of
marker gene transcripts. The heterogeneity of marker gene
expression levels in NSCLC cells limits the reliability of an assay
method with a single-marker detection scheme. Several literature
reports have described PCR methods for the detection of tumor cells
dispersed in the circulation. However, not one tumor marker is
consistently and specifically expressed in all of the primary
tumors of a particular malignancy. Literature reports have also
shown that a panel of marker genes provides a more reliable and
informative approach than a single-marker assay for the detection
of melanoma and breast cancer cells in blood. Such assays for lung
cancer have been limited by the availability of molecular
markers.
[0007] The presence of epithelial cancer cells in the bone marrow
and in the peripheral blood of patients with carcinoma has been
reported in literature reports and prior arts. In contrast to bone
marrow aspirates, peripheral blood samples can be obtained
routinely and more readily. Carcinoma accounts for around 85% of
human cancers and the carcinoma cells are of epithelial cell
lineage. Techniques such as immunocytology and flow cytometry have
been employed in prior arts to detect circulating cancer cells in
the peripheral blood. However, both techniques are based on
extracting or labeling intact carcinoma cells in circulation by
antibodies targeting specifically to the epithelial cell surface
antigens such as EpCAM and others. Malignant carcinoma cancer cells
often are de-differentiated and lose the characteristic epithelial
cell surface antigens. In addition, it is known in cancer research
field that EpCAM gene expression is often suppressed to facilitate
tumor metastasis. Therefore, the antibody based detection methods
have been reported to have low positive detection rates or high
false negative rates. Polymerase chain reaction (PCR) has been
employed to detect disseminated tumor cells in peripheral blood.
Several literature reports have described the use of PCR for
detecting circulating cancer cells in the peripheral blood of
patients of various cancers. For instance, Peck et al., reported
the use of cytokeratin 19 as the maker gene for detecting
circulating cancer cells in NSCLC patients with an overall positive
detection rate around 40%.
[0008] Compared with immunocytology and flow cytometry, PCR has the
advantages that it is more readily available, less involved in the
operating procedures, less instrument cost, and others. On the
other hand, PCR is not able to yield the number of counts of
circulating cancer cell in a sample like the other two
techniques.
[0009] To overcome the current technology difficulties in achieving
high positive detection rate and rapid assessment of lung cancer
therapy efficacy and relapse detection, a panel of marker genes for
achieving high positive detection rate by qPCR and a quantitative
analysis method for predicting lung cancer treatment outcome and
for prognosis are needed.
SUMMARY OF THE INVENTION
[0010] The present invention fulfills the needs in lung cancer
treatment by teaching a rapid efficacy assessment method for lung
cancer therapy and relapse detection.
[0011] The purpose of the present invention is to teach an
assessment method for lung cancer therapy. More especially, it
teaches a rapid efficacy assessment method for lung cancer therapy
by identifying and employing a panel of marker genes for real-time
quantitative PCR (qPCR) assay to quantitatively measure the amount
of circulating lung cancer cells in body fluids.
[0012] Another purpose of the present method is to teach a method
for cancer relapse detection by using real time qPCR with a panel
of marker genes for detecting circulating lung cancer cells in body
fluids.
[0013] The present invention identifies a panel of markers for the
detection of circulating cancer cells in NSCLC patients by in
silico analysis of the National Cancer Institute-Cancer Genome
Anatomy Project database. The present invention also teaches a
quantitative analysis method to calculate load of cancer cells in
the circulation. The quantitative analysis method yields results
that are highly correlated with the treatment outcomes of lung
cancer patients and serves to predict the treatment outcome in a
short time after the treatment is administered.
[0014] The method of assessing lung cancer therapy comprises:
collecting a body fluid from a subject, extracting total RNA of the
body fluid sample, employing qPCR to amplify marker gene
transcripts of total RNA for detecting cancer cells in body fluid,
and analyzing qPCR threshold cycle number with a set of
mathematical formulae.
[0015] The present invention further teaches a method to translate
expression level of multiple gene transcripts measured by qPCR to
the amount of circulating lung cancer cells which is termed cancer
cell load (Lc) in this invention.
[0016] The present invention further teaches a scoring method and
mathematical formulae for calculating cancer cell load, Lc, and
predicting lung cancer treatment outcome with the Lc value.
[0017] The present invention now will be described more fully
hereinafter with reference to the accompanying drawings, which are
intended to be read in conjunction with both this summary, the
detailed description and any preferred and/or particular
embodiments specifically discussed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The present invention will become apparent upon reading the
following detailed description of the present invention in
conjunction with the drawings, as follows:
[0019] FIG. 1 shows the analysis of positive detection rates with a
panel of 4 marker genes. A, positive detection rate for the
individual four marker genes. B, positive detection rate increases
with the number of marker genes used in the assay.
[0020] FIG. 2 shows the correlation of Lc value with the
concentration of cancer cell number in peripheral blood. A, plot to
show that Lc value is linearly correlated with the number of
circulating cancer cells. Inset, Lc value versus the number of
cancer cells between 0 and 480 cells/mL of peripheral blood. B,
median survival time for patients of various TNM stages and with
high (.gtoreq.1) or low (<1) Lc values. Inset, survival time of
patient classified by the TNM staging method. Bars, standard
deviation (SD).
[0021] FIG. 3 shows the survival analysis of late stage patients
with high (.gtoreq.1) or low (<1) Lc values. A, Kaplan-Meier
survival plots of late TNM stage patients (IIIb and IV). B,
Kaplan-Meier survival plots for the same late stage patients
grouped by the Lc value. C, Kaplan-Meier survival plots for the
stage IIIb patients grouped by the Lc value. D, Kaplan-Meier
survival plots for the stage IV patients grouped by the Lc
value.
[0022] FIG. 4 shows lung cancer therapy efficacy assessment with
circulating cancer cell detection for six different NSCLC patients.
Median survival time of each patient is indicated in the figure
panels. The designations for the treatment and sampling day are
described in the text.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention is directed to a method for lung
cancer therapy assessment and a method of cancer relapse detection.
Unlike current imaging assessment methods, the present invention
provides a rapid assessment that uses multiple marker genes in qPCR
assay for detecting circulating lung cancer cells in body
fluids.
[0024] Compared with immunohistology and flow cytometry assessment
methods, the present invention requires no additional antibody
antigen interaction process. Extra molecular recognition process
reduces the detection rate. Compared with the RT-PCR detection
method for circulating cancer cell in prior art, using a panel of
marker genes instead of a single marker gene improves the detection
rate. On the other hand, it is not trivial to quantitatively
analyze and integrate the expression level of multiple transcripts
in a qPCR assay and correlating the analysis results to predict
treatment outcome. The present invention teaches a set of
mathematical formulae which yield results well correlated with lung
cancer treatment outcome.
[0025] The multiple marker genes that may be selected include, but
are not limited to, keratin 19 (KRT19), ubiquitin thiolesterase
(UCHL1), fibronectin 1 (FN1), and tripartite motif-containing 28
(TRIM28).
[0026] The method of the present invention for rapid lung cancer
therapy efficacy assessment comprises the following steps:
[0027] collecting a body fluid sample from a human subject;
[0028] extracting total RNA from said body fluid sample;
[0029] amplifying a panel of gene transcripts of said total RNA by
qPCR;
[0030] measuring the expression level of each gene transcript in
said panel of gene transcripts of said total RNA from a number of
healthy controls and lung cancer patients, wherein a qPCR threshold
cycle number is used to represent the expression level of the gene
transcripts;
[0031] deriving a reference threshold score using the qPCR
threshold cycle numbers of the gene transcripts in the panel
measured for healthy controls and lung cancer patients;
[0032] calculating an indicative score using the qPCR threshold
cycle numbers of the gene transcripts in the panel measured for a
lung cancer patient both before and after therapy; and
[0033] determining therapy efficacy by comparing the indicative
scores obtained before and after therapy.
[0034] The formulae used in this invention to calculate the values
in these steps can be found in the section "Quantitative Analysis
(Scoring) of the PCR Results" below.
[0035] In the present invention, the body fluids are collected
from, but not limit to, peripheral blood or pleural effusion.
[0036] In the present invention, the number of multiple gene
transcripts selected for amplification is more than two.
[0037] In the present invention, the nucleic acid extraction is
done without prior antibody/antigen interaction or other molecular
recognition processes to isolate cancer cells from normal blood
cells.
[0038] In the present invention, the quantitative analysis for
therapy efficacy is performed as early as one day after the
therapeutic regimen is administered.
[0039] The method of the present invention for cancer relapse
detection comprises the following steps:
[0040] collecting a body fluid sample from a human subject;
[0041] extracting total RNA from said body fluid sample;
[0042] amplifying a panel of gene transcripts of said total RNA by
qPCR;
[0043] measuring the expression level of each gene transcript in
said panel of gene transcripts of said total RNA from a number of
healthy controls and lung cancer patients, wherein a qPCR threshold
cycle number is used to represent the expression level of the gene
transcripts;
[0044] deriving a reference threshold score using the qPCR
threshold cycle numbers of the gene transcripts in the panel
measured for healthy controls and lung cancer patients;
[0045] calculating an indicative score using the qPCR threshold
cycle numbers of the gene transcripts in the panel measured for a
lung cancer patient; and
[0046] determining the presence of circulating lung cancer cells by
comparing the indicative scores with the reference threshold
score.
[0047] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The practice of the
present invention will employ, unless otherwise indicated,
conventional techniques of cell biology, molecular biology, and
biochemistry which are within the skill of the art. Such techniques
are explained fully in the literature. The following examples are,
therefore, to be considered as merely illustrative, and not
limitative of the remainder of the disclosure in any way.
[0048] Materials and Methods
[0049] Patients and Specimens
[0050] Peripheral blood samples were obtained with informed consent
from 54 patients with histologically documented NSCLC in the
National Taiwan University Hospital. Staging procedures included
chest radiography, bronchoscopy, brain and thoracic computed
tomography, sonography, and bone scintigraphy. The follow-up period
of the lung cancer patients was up to 85 months for stage I
patients who were still alive in May 2003. The normal control
population consisted of 24 healthy volunteers with no history or
present diagnosis of malignancy. Among the 54 patients, 32 were men
and 22 were women with median age of 65 years (SD=11, range=28-81).
In the healthy control group, 16 were men and 8 were women with
median age of 57.5 years (SD=15.1, range=27-78). Adenocarcinoma
lung cancer cell lines (A549, CL3, H928, CL1-0, CL1-5, CRL-5865,
CRL-5806, and CRL-5807) and squamous carcinoma lung cancer cell
lines (NCI-H520, H2981, CRL-5802, and HTB-54) were used to validate
the candidate markers and for analyzing the correlation between Lc
value and cancer cell number.
[0051] Sample Collection and RNA Preparation
[0052] The blood sample collection and RNA preparation methods were
the same as described by Peck et al. (Peck, Cancer Res.
58:2761-2765, 1998) Briefly, two samples were collected from each
subject with Vacutainers (Becton Dickinson, Rutherford, N.J.). The
first tube with 1 to 2 mL of peripheral blood was discarded and
only the second tube with 3 to 4 mL of blood was assayed to avoid
epithelial cell contamination by the needle when it pierced through
the skin. Total RNA was extracted with the QIAamp RNA Blood Mini
kit (Qiagen, Hiden, Germany) within 2 hours after the blood samples
were collected.
[0053] Identification of Candidate Marker Genes
[0054] To take advantage of the vast information of the expressed
sequence tags databases generated with cancer cell lines, we used
the cDNA Digital Gene Expression Displayer developed by the Cancer
Genome Anatomy Project (Strausberg, J. Pathol, 195:31-40, 2001) to
identify genes that were differentially expressed between lung
cancer cells and leukocytes. The Digital Gene Expression Displayer
program identified differentially expressed genes among 47,036
sequences in five lung cancer cDNA libraries and 21,460 sequences
in six leukocyte cDNA libraries with a P filter set at 0.01. The
differentially expressed genes were ranked by sequence odds ratio.
The genes with the highest sequence odds ratios were selected as
candidate marker genes for quantitative PCR (qPCR) assay. The in
silico Digital Gene Expression Displayer program search of the
National Cancer Institute-Cancer Genome Anatomy Project database
yielded 85 overexpressed genes with a sequence odds ratio >16
between the lung cancer cDNA libraries and the leukocyte cDNA
libraries. These candidate genes were further verified by real-time
quantitative PCR (qPCR). All of the cancer cell lines are listed as
above and pooled and peripheral blood mononuclear cells from 12
healthy controls were used as samples in the first round
verification. Fifty-nine candidate marker genes showed >2-fold
differential expression ratios, but only 19 genes had differential
expression ratios >100,000. Marker genes with large differential
expression ratios are required to detect rare circulating cancer
cells in blood samples containing millions of peripheral blood
mononuclear cells. By using qPCR to detect the presence of 19
candidate genes in the clinical specimens of 54 NSCLC patients and
24 normal controls, four marker genes including KRT19 were
identified to show positive detection in at least two NSCLC
patients. The four marker genes are listed in Table 1.
[0055] The first three marker genes had negligible expression in
the blood samples of normal controls by the qPCR assay and are
labeled as genes A to C. The fourth gene, labeled as gene D, had
high expression in the cancer patient group versus the normal
control group, but had residual expression in normal peripheral
blood mononuclear cells. Therefore, the present invention sets the
positive detection threshold at 99% confidence interval in the
normal control group to avoid false positive detection.
TABLE-US-00001 TABLE 1 List of a panel of four marker genes Gene
Title RefSeq no. GeneID A keratin 19 (KRT19) NM_002276 3880 B
ubiquitin thiolesterase (UCHL1) NM_004181 7345 C fibronectin 1
(FN1) NM_054034 2335 D tripartite motif-containing 28 (TRIM28)
NM_005762 10155
[0056] Quantitative PCR Assay
[0057] To detect a few cancer cells in the circulation, a highly
sensitive PCR amplification is necessary. cDNA was derived from 1
to 2 .mu.g of total RNA by random primed reverse transcription, and
nested PCR was used to amplify the candidate marker gene
transcripts for detection. The primer sets for qPCR amplification
are listed in Table 1 of U.S. Provisional Application No.
60/596,104, filed Sep. 1, 2005, from which priority benefit is
claimed by this application and which is incorporated herein. The
first round (outer) of the nested PCR was done using 1 .mu.L of
20-fold diluted cDNA with a PCR mixture containing 0.1 .mu.mol/L
outer primer pair, 0.2 mmol/L deoxynucleotide triphosphate, 50
mmol/L Tris-HCl (pH 8.3), 10 mmol/L KCl, 5 mmol/L
(NH.sub.4).sub.2SO.sub.4, 2 mmol/L MgCl.sub.2, and 0.75 units of
FastStart Taq DNA polymerase (Roche, Mannheim, Germany) in a total
volume of 12.5 .mu.L. The PCR conditions were one cycle at
94.degree. C. for 7 minutes followed by 25 cycles at 94.degree. C.
for 50 seconds, 60.degree. C. for 50 seconds, 72.degree. C. for 35
seconds, and a final extension at 72.degree. C. for 10 minutes.
[0058] For the second round (inner) of the nested PCR
amplification, quantitative measurement was performed with qPCR
assay. The reaction mixture contained 2 .mu.L of the first round
PCR product, 0.25 .mu.mol/L inner primers, and SYBR Green PCR
master mix (Applied Biosystems, Foster City, Calif.) in a total
volume of 20 .mu.L. The qPCR assays were done with an ABI prism
7000 SDS (Applied Biosystems) instrument. The qPCR condition was
95.degree. C. for 10 minutes followed by 40 cycles at 95.degree. C.
for 15 seconds, 60.degree. C. for 25 seconds, and 72.degree. C. for
35 seconds.
[0059] Quantitative Analysis (Scoring) of the PCR Results
[0060] By using a qPCR instrument, the threshold cycle (C.sub.T),
the fractional cycle number at which the SYBR Green I fluorescence
exceeded a set level above baseline, was determined. We used GAPDH
mRNA as an internal control. The relative amount of mRNA,
normalized against the GAPDH mRNA, was expressed as
C.sub.T=C.sub.T.sup.(GAPDH)-C.sub.T.sup.(marker gene). If the
fluorescence signal was undetected after 40 cycles, the C.sub.T
value was given the maximum cycle number of 40 for analysis
convenience. The differential expression ratio of a candidate
marker gene, Q, for patients versus normal controls was calculated
by Q=2.sup.mean of .sup.in normal. To estimate the number of
circulating cancer cells, we normalized the differential expression
ratio of each marker gene to take into account the different amount
of the marker gene transcripts in cancer cells. We then summed up
the expression ratios of the marker genes to estimate the cancer
cell load in the circulation in a semi-quantitative way. Cancer
cells and their gene expression profiles are heterogeneous in
individuals. To take the heterogeneity of gene expression in
different patients into account, the expression of marker genes
need to be normalized among test subjects. The formula for
normalizing the expression ratio, E.sub.ij, of a marker gene is
E.sub.ij=(Q.sub.ij-Q.sub.j)/.sigma..sub.j, where i is the patient
index, j is the marker gene index, Q.sub.ij is the differential
expression ratio of marker gene j in patient i, Q.sub.j is the mean
and .sigma..sub.j is the standard deviation (SD) of the expression
ratios of the 54 patients for marker gene j. The load of cancer
cells, Lc, in the circulation of a patient is defined as
Lc=.SIGMA.E.sub.j where n is the number of marker genes. In this
study, the load of cancer cell, Lc, is used as an indicative score
for the amount of circulating cancer cells. The value of Lc ranged
between -2.2 and 8.4.
[0061] Statistical Analysis
[0062] Fisher's exact test and Student's t test were used to
compare the clinicopathologic characteristics of patients with low
and high Lc values. All statistical tests were two sided. Survival
time of the patients was calculated from the day of specimen
collection. Survival curves were obtained by the Kaplan-Meier
method. The difference of survival times between two groups was
analyzed with the log-rank test. P<0.05 was considered
statistically significant.
Embodiment 1
Enhancement of Positive Detection Rate with Multiple Marker
Genes
[0063] As shown in FIG. 1A, the positive detection rate of
circulating cancer cells in NSCLC patients was 41%, 11%, 39%, and
11% for genes A to D, respectively. The fractions of positives
among all the patients are indicated in the columns.
[0064] Increasing the number of marker genes raised the positive
detection rate for NSCLC patients to 72% (39 of 54) compared with
41% (22 of 54) positive detection rate using the KRT19 marker gene
alone (FIG. 1B). These results prove that using multiple markers
indeed improves the positive detection rate. The B marker gene was
not detected in squamous cell lung cancer patients, but the other
three genes were detected in patients with adenocarcinoma or
squamous cell carcinoma.
Embodiment 2
Circulating Cancer Cell Load and Patient Outcome
[0065] To investigate the correlation between the number of
circulating cancer cells and patient outcome, the present invention
used cancer cell load Lc (see MATERIALS AND METHODS) as a measure
to indicate the number of cancer cells in circulation.
[0066] By definition, Lc increases with the number of circulating
cells. The correlation between the number of circulating cancer
cells and Lc values can be determined by spiking different numbers
of lung cancer cells (CL1-0) into peripheral blood mononuclear
cells (FIG. 2A). The present invention shows a good correlation
(r=0.99) between cancer cell number and Lc value.
[0067] The data shown in FIGS. 2B and 3 indicate that patients of
the same stage who had higher Lc had worse outcomes. The results
indicate that Lc measurement is a supplementary tool to the
traditional TNM staging method to better predict the outcome of
cancer patients.
[0068] To determine whether Lc can be used as an indicator of
patient outcome, the present invention analyzed the Lc value versus
the survival time of stage I to IIIa patients who received surgical
resection. We found that patients who survived for more than 5
years had Lc values less than 1. Therefore, we set Lc=1 as the
reference threshold score for the subsequent prognosis studies in
the 54 lung cancer patients. The study divided the patients at
different stages into two groups (Lc.gtoreq.1 or Lc<1) and
examined their survival time. As expected, late-stage patients had
shorter survival times than did early-stage patients (FIG. 2B,
inset). The cancer load (Lc) study further distinguished that for
patients of the same stage, those with low Lc had longer survival
time than those with high Lc value (FIG. 2B). For the operable
early-stage patients, the survival time is highly correlated
(P=0.002) with Lc value. The Lc value was then used to analyze the
prognosis of late-stage patients (stages IIIb and IV) with
Kaplan-Meier survival plots. The survival time difference was more
significant by using Lc as a classification parameter (FIG. 3B,
P=0.006) than by using the traditional TNM classification (FIG. 3A,
P=0.03). The number of patients in each group is shown in
parentheses in the figures. We further used the Lc parameter to
divide the patients of the same stage into low (Lc<1) and high
(Lc.gtoreq.1) value groups and examined the survival plots. The
survival time of stage IIIb patients with low and high Lc value was
significantly different (FIG. 3C, P=0.026). The same analysis on
stage IV patients did not achieve statistical significance (FIG.
3D, P=0.09). These prognosis studies establish using Lc=1 as the
reference threshold score for detecting the presence of circulating
lung cancer cells.
[0069] Using the four marker genes and setting the reference
threshold score to 1, the positive detection rates of patients with
circulating cancer cells were 67% (4 of 6) for stage I, 100% (4 of
4) for stage II, 67% (6 of 9) for stage IIIa, 67% (14 of 21) for
stage IIIb, and 79% (11 of 14) for stage IV. The detection rate
indicates the sensitivity of using the four markers for detecting
the presence of circulating NSCLC cells.
Embodiment 3
Assessment of Therapy Efficacy
[0070] FIG. 4 shows the assessment of six patients before and after
therapy. The stage I, II, and IIIa patients received surgical
resection, whereas the stage IIIb and IV patients received
chemotherapy. "d-n" indicates that the samples were collected n
days before therapy, "dn" indicates that the samples were collected
on the nth day after therapy, and "n/" indicates the course number
of chemotherapy. The stage I patient had negative Lc values before
and after treatment and was still alive on the last follow-up date
(72.3 months). The Lc values decreased for the stage II and IIIb
patients after therapy. The stage IIIa and IV patients had higher
Lc values after the last course of treatment and had short survival
times. These results suggest that measurement of the cancer cell
load, Lc, can be used to reveal whether therapy is efficacious.
Embodiment 4
Relapse Detection
[0071] In FIG. 4 the stage IIIb patient's treatment was efficacious
and the Lc value decreased after the first treatment. However, the
Lc value went back up to exceed 1 and the patient was given a
second course of treatment on the next day. The second treatment
for the stage IIIb was assessed to be effective by the method
disclosed in this invention on the fifth day after the treatment.
The survival time was 60.8 months for the patient. The last example
in FIG. 4 was a stage IV patient. The Lc value decreased on the
second day of the treatment. The Lc value of the patient went up
again to exceed 1 in a follow-up examination and a second treatment
was given on the next day. However, the second treatment failed to
completely eradicate the cancer cells and the Lc value kept rising
with time. The patient had a survival time of 8.6 months.
TABLE-US-00002 TABLE 2 Clinicopathologic characteristics and their
correlation with Lc value of NSCLC patients Lc Characteristic Low
High P Age (y), mean .+-. SD 63.6 .+-. 10.5 61.3 .+-. 13.0 0.549*
Gender, no. patients Male 20 20 0.03 Female 20 2 Smoking, no.
patients No 24 7 0.546 Yes 16 7 Histology, no. patients
Adenocarcinoma 25 10 0.442 Squamous cell 12 2 carcinoma Poorly
differentiated 3 2 Stage.dagger. I-IIIa 14 5 1.0 IIIb-IV 26 9
*Derived with Student's t test; other Ps were derived with Fisher's
exact test. All statistical tests were two-sided. .dagger.Tumor
stage was classified according to the International System for
Staging Lung Cancer.
[0072] The percentage of patients with high Lc value is greater for
the late-stage patients than for the early-stage patients except
for the stage II patients. The invention study looked into this
issue and found that the markers have different detection rates for
different histologic types of NSCLC. The markers are more sensitive
for detecting squamous carcinoma (85.7%; 12 of 14) compared with
adenocarcinomas (68.6%; 24 of 35) and others (60%, 3 of 5). An
investigation on the histologic types of the NSCLC patients
revealed that the available stage II patients were composed of
three (75%) squamous carcinoma and one (25%) adenocarcinoma
patients, whereas the other stage patients were composed of 11% to
33% squamous carcinoma, 50% to 71% adenocarcinoma, and 5% to 22%
poorly differentiated cell type patients. The higher detection rate
of the stage II patients can therefore be attributed to the limited
number of clinical samples and their histologic composition.
[0073] The data shows that detection of circulating cancer cells is
a valid supplement to the TNM method for better cancer staging. The
two methods combined together provide better information for
designing lung cancer treatment strategies. In the study of this
invention, the 5-year survival rate of early-stage (I-IIIa) NSCLC
patients was 30% to 50% after surgical resection. This invention
teaches a more precise staging method by including detection of
circulating cancer cells to aid in deciding whether adjuvant
therapeutic regimens in addition to tumor resection are beneficial
to the patients.
[0074] At present, a reliable serologic biomarker assay for
assessing the treatment response of NSCLC patients is not
available. Two courses of chemotherapy are traditionally given
before imaging is done to evaluate the treatment response of NSCLC
patients. It showed in lung cancer patients that the Lc could be
used for monitoring therapeutic response and relapse. Because
different cancer cell types have different levels of marker gene
expression, patients with higher Lc values do not necessarily have
more circulating cancer cells in their peripheral blood than do
patients with lower Lc values. Nevertheless, the semiquantitative
approach is useful for measuring the relative cancer cell load in a
patient's peripheral blood to monitor the effectiveness of
treatment. The present method highlights an alternative approach to
rapidly assess the treatment response of NSCLC patients. Compared
with imaging methods which take weeks to detect the change of tumor
size for therapy efficacy assessment, the invention teaches a
method capable of assessing therapy efficacy on the next day of
treatment as shown in FIG. 4. The method of the present invention
may therefore help to design more comprehensive and reasonable
therapeutic regimens at earlier dates for NSCLC patients.
[0075] Unless defined otherwise, the meanings of all technical and
scientific terms used herein are those commonly understood by one
of ordinary skill in the art to which this invention belongs. One
of ordinary skill in the art will also appreciate that any methods
and materials similar or equivalent to those described herein can
also be used to practice or test the invention. Further, all
publications mentioned herein are incorporated by reference.
[0076] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims.
Sequence CWU 1
1
411513DNAHomo sapiens 1cgcccctgac accattcctc ccttcccccc tccaccggcc
gcgggcataa aaggcgccag 60gtgagggcct cgccgctcct cccgcgaatc gcagcttctg
agaccagggt tgctccgtcc 120gtgctccgcc tcgccatgac ttcctacagc
tatcgccagt cgtcggccac gtcgtccttc 180ggaggcctgg gcggcggctc
cgtgcgtttt gggccggggg tcgcctttcg cgcgcccagc 240attcacgggg
gctccggcgg ccgcggcgta tccgtgtcct ccgcccgctt tgtgtcctcg
300tcctcctcgg gggcctacgg cggcggctac ggcggcgtcc tgaccgcgtc
cgacgggctg 360ctggcgggca acgagaagct aaccatgcag aacctcaacg
accgcctggc ctcctacctg 420gacaaggtgc gcgccctgga ggcggccaac
ggcgagctag aggtgaagat ccgcgactgg 480taccagaagc aggggcctgg
gccctcccgc gactacagcc actactacac gaccatccag 540gacctgcggg
acaagattct tggtgccacc attgagaact ccaggattgt cctgcagatc
600gacaatgccc gtctggctgc agatgacttc cgaaccaagt ttgagacgga
acaggctctg 660cgcatgagcg tggaggccga catcaacggc ctgcgcaggg
tgctggatga gctgaccctg 720gccaggaccg acctggagat gcagatcgaa
ggcctgaagg aagagctggc ctacctgaag 780aagaaccatg aggaggaaat
cagtacgctg aggggccaag tgggaggcca ggtcagtgtg 840gaggtggatt
ccgctccggg caccgatctc gccaagatcc tgagtgacat gcgaagccaa
900tatgaggtca tggccgagca gaaccggaag gatgctgaag cctggttcac
cagccggact 960gaagaattga accgggaggt cgctggccac acggagcagc
tccagatgag caggtccgag 1020gttactgacc tgcggcgcac ccttcagggt
cttgagattg agctgcagtc acagctgagc 1080atgaaagctg ccttggaaga
cacactggca gaaacggagg cgcgctttgg agcccagctg 1140gcgcatatcc
aggcgctgat cagcggtatt gaagcccagc tgggcgatgt gcgagctgat
1200agtgagcggc agaatcagga gtaccagcgg ctcatggaca tcaagtcgcg
gctggagcag 1260gagattgcca cctaccgcag cctgctcgag ggacaggaag
atcactacaa caatttgtct 1320gcctccaagg tcctctgagg cagcaggctc
tggggcttct gctgtccttt ggagggtgtc 1380ttctgggtag agggatggga
aggaagggac ccttaccccc ggctcttctc ctgacctgcc 1440aataaaaatt
tatggtccaa gggaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1500aaaaaaaaaa aaa 151321110DNAHomo sapiens 2cctgggcggc tccgctagct
gtttttcgtc ttccctaggc tatttctgcc gggcgctccg 60cgaagatgca gctcaagccg
atggagatca accccgagat gctgaacaaa gtgctgtccc 120ggctgggggt
cgccggccag tggcgcttcg tggacgtgct ggggctggaa gaggagtctc
180tgggctcggt gccagcgcct gcctgcgcgc tgctgctgct gtttcccctc
acggcccagc 240atgagaactt caggaaaaag cagattgaag agctgaaggg
acaagaagtt agtcctaaag 300tgtacttcat gaagcagacc attgggaatt
cctgtggcac aatcggactt attcacgcag 360tggccaataa tcaagacaaa
ctgggatttg aggatggatc agttctgaaa cagtttcttt 420ctgaaacaga
gaaaatgtcc cctgaagaca gagcaaaatg ctttgaaaag aatgaggcca
480tacaggcagc ccatgatgcc gtggcacagg aaggccaatg tcgggtagat
gacaaggtga 540atttccattt tattctgttt aacaacgtgg atggccacct
ctatgaactt gatggacgaa 600tgccttttcc ggtgaaccat ggcgccagtt
cagaggacac cctgctgaag gacgctgcca 660aggtctgcag agaattcacc
gagcgtgagc aaggagaagt ccgcttctct gccgtggctc 720tctgcaaggc
agcctaatgc tctgtgggag ggactttgct gatttcccct cttcccttca
780acatgaaaat atataccccc ccatgcagtc taaaatgctt cagtacttgt
gaaacacagc 840tgttcttctg ttctgcagac acgccttccc ctcagccaca
cccaggcact taagcacaag 900cagagtgcac agctgtccac tgggccattg
tggtgtgagc ttcagatggt gaagcattct 960ccccagtgta tgtcttgtat
ccgatatcta acgctttaaa tggctacttt ggtttctgtc 1020tgtaagttaa
gaccttggat gtggtttaat tgtttgtcct caaaaggaat aaaacttttc
1080tgctgataag ataaaaaaaa aaaaaaaaaa 111032402DNAHomo sapiens
3gcccgcgccg gctgtgctgc acagggggag gagagggaac cccaggcgcg agcgggaaga
60ggggacctgc agccacaact tctctggtcc tctgcatccc ttctgtccct ccacccgtcc
120ccttccccac cctctggccc ccaccttctt ggaggcgaca acccccggga
ggcattagaa 180gggatttttc ccgcaggttg cgaagggaag caaacttggt
ggcaacttgc ctcccggtgc 240gggcgtctct cccccaccgt ctcaacatgc
ttaggggtcc ggggcccggg ctgctgctgc 300tggccgtcca gtgcctgggg
acagcggtgc cctccacggg agcctcgaag agcaagaggc 360aggctcagca
aatggttcag ccccagtccc cggtggctgt cagtcaaagc aagcccggtt
420gttatgacaa tggaaaacac tatcagataa atcaacagtg ggagcggacc
tacctaggca 480atgcgttggt ttgtacttgt tatggaggaa gccgaggttt
taactgcgag agtaaacctg 540aagctgaaga gacttgcttt gacaagtaca
ctgggaacac ttaccgagtg ggtgacactt 600atgagcgtcc taaagactcc
atgatctggg actgtacctg catcggggct gggcgaggga 660gaataagctg
taccatcgca aaccgctgcc atgaaggggg tcagtcctac aagattggtg
720acacctggag gagaccacat gagactggtg gttacatgtt agagtgtgtg
tgtcttggta 780atggaaaagg agaatggacc tgcaagccca tagctgagaa
gtgttttgat catgctgctg 840ggacttccta tgtggtcgga gaaacgtggg
agaagcccta ccaaggctgg atgatggtag 900attgtacttg cctgggagaa
ggcagcggac gcatcacttg cacttctaga aatagatgca 960acgatcagga
cacaaggaca tcctatagaa ttggagacac ctggagcaag aaggataatc
1020gaggaaacct gctccagtgc atctgcacag gcaacggccg aggagagtgg
aagtgtgaga 1080ggcacacctc tgtgcagacc acatcgagcg gatctggccc
cttcaccgat gttcgtgcag 1140ctgtttacca accgcagcct cacccccagc
ctcctcccta tggccactgt gtcacagaca 1200gtggtgtggt ctactctgtg
gggatgcagt ggctgaagac acaaggaaat aagcaaatgc 1260tttgcacgtg
cctgggcaac ggagtcagct gccaagagac agctgtaacc cagacttacg
1320gtggcaactc aaatggagag ccatgtgtct taccattcac ctacaatggc
aggacgttct 1380actcctgcac cacagaaggg cgacaggacg gacatctttg
gtgcagcaca acttcgaatt 1440atgagcagga ccagaaatac tctttctgca
cagaccacac tgttttggtt cagactcgag 1500gaggaaattc caatggtgcc
ttgtgccact tccccttcct atacaacaac cacaattaca 1560ctgattgcac
ttctgagggc agaagagaca acatgaagtg gtgtgggacc acacagaact
1620atgatgccga ccagaagttt gggttctgcc ccatggctgc ccacgaggaa
atctgcacaa 1680ccaatgaagg ggtcatgtac cgcattggag atcagtggga
taagcagcat gacatgggtc 1740acatgatgag gtgcacgtgt gttgggaatg
gtcgtgggga atggacatgc attgcctact 1800cgcagcttcg agatcagtgc
attgttgatg acatcactta caatgtgaac gacacattcc 1860acaagcgtca
tgaagagggg cacatgctga actgtacatg cttcggtcag ggtcggggca
1920ggtggaagtg tgatcccgtc gaccaatgcc aggattcaga gactgggacg
ttttatcaaa 1980ttggagattc atgggagaag tatgtgcatg gtgtcagata
ccagtgctac tgctatggcc 2040gtggcattgg ggagtggcat tgccaacctt
tacagaccta tccaagctca agtggtcctg 2100tcgaagtatt tatcactgag
actccgagtc agcccaactc ccaccccatc cagtggaatg 2160caccacagcc
atctcacatt tccaagtaca ttctcaggtg gagacctgtg agtatcccac
2220ccagaaacct tggatactga gtctcctaat cttatcaatt ctgatggttt
ctttttttcc 2280cagcttttga gccaacaact ctgattaact attcctatag
catttactat atttgtttag 2340tgaacaaaca atatgtggtc aattaaattg
acttgtagac tgaaaaaaaa aaaaaaaaaa 2400aa 240242989DNAHomo sapiens
4ggcgcgcggg cgagcggttg tgcttgtgct tgtggcgcgt ggtgcgggtt tcggcggcgg
60ctgaggaaga agcgcgggcg gcgccttcgg gaggcgagca ggcagcagtt ggccgtgccg
120tagcagcgtc ccgcgcgcgg cgggcagcgg cccaggaggc gcgtggcggc
gctcggcctc 180gcggcggcgg cggcggcagc ggcccagcag ttggcggcga
gcgcgtctgc gcctgcgcgg 240cgggccccgc gcccctcctc cccccctggg
cgcccccggc ggcgtgtgaa tggcggcctc 300cgcggcggca gcctcggcag
cagcggcctc ggccgcctct ggcagcccgg gcccgggcga 360gggctccgct
ggcggcgaaa agcgctccac cgccccttcg gccgcagcct cggcctctgc
420ctcagccgcg gcgtcgtcgc ccgcgggggg cggcgccgag gcgctggagc
tgctggagca 480ctgcggcgtg tgcagagagc gcctgcgacc cgagagggag
ccccgcctgc tgccctgttt 540gcactcggcc tgtagtgcct gcttagggcc
cgcggccccc gccgccgcca acagctcggg 600ggacggcggg gcggcgggcg
acggcaccgt ggtggactgt cccgtgtgca agcaacagtg 660cttctccaaa
gacatcgtgg agaattattt catgcgtgat agtggcagca aggctgccac
720cgacgcccag gatgcgaacc agtgctgcac tagctgtgag gataatgccc
cagccaccag 780ctactgtgtg gagtgctcgg agcctctgtg tgagacctgt
gtagaggcgc accagcgggt 840gaagtacacc aaggaccata ctgtgcgctc
tactgggcca gccaagtctc gggatggtga 900acgtactgtc tattgcaacg
tacacaagca tgaacccctt gtgctgtttt gtgagagctg 960tgatactctc
acctgccgag actgccagct caatgcccac aaggaccacc agtaccagtt
1020cttagaggat gcagtgagga accagcgcaa gctcctggcc tcactggtga
agcgccttgg 1080ggacaaacat gcaacattgc agaagagcac caaggaggtt
cgcagctcaa tccgccaggt 1140gtctgacgta cagaagcgtg tgcaagtgga
tgtcaagatg gccatcctgc agatcatgaa 1200ggagctgaat aagcggggcc
gtgtgctggt caatgatgcc cagaaggtga ctgaggggca 1260gcaggagcgc
ctggagcggc agcactggac catgaccaag atccagaagc accaggagca
1320cattctgcgc tttgcctctt gggctctgga gagtgacaac aacacagccc
ttttgctttc 1380taagaagttg atctacttcc agctgcaccg ggccctcaag
atgattgtgg atcccgtgga 1440gccacatggc gagatgaagt ttcagtggga
cctcaatgcc tggaccaaga gtgccgaggc 1500ctttggcaag attgtggcag
agcgtcctgg cactaactca acaggccctg cacccatggc 1560ccctccaaga
gccccagggc ccctgagcaa gcagggctct ggcagcagcc agcccatgga
1620ggtgcaggaa ggctatggct ttgggtcagg agatgatccc tactcaagtg
cagagcccca 1680tgtgtcaggt gtgaaacggt cccgctcagg tgagggcgag
gtgagcggcc ttatgcgcaa 1740ggtgccacga gtgagccttg aacgcctgga
cctggacctc acagctgaca gccagccacc 1800cgtcttcaag gtcttcccag
gcagtaccac tgaggactac aaccttattg ttattgaacg 1860tggcgctgcc
gctgcagcta ccggccagcc agggactgcg cctgcaggaa cccctggtgc
1920cccacccctg gctggcatgg ccattgtcaa ggaggaggag acggaggctg
ccattggagc 1980ccctcctact gccactgagg gccctgagac caaacctgtg
cttatggctc ttgcggaggg 2040tcctggtgct gagggtcccc gcctggcctc
acctagtggc agcaccagct cagggctgga 2100ggtggtggct cctgagggta
cctcagcccc aggtggtggc ccgggaaccc tggatgacag 2160tgccaccatt
tgccgtgtct gccagaagcc aggcgatctg gttatgtgca accagtgtga
2220gttttgtttc cacctggact gtcacctgcc ggccctgcag gatgtaccag
gggaggagtg 2280gagctgctca ctctgccatg tgctccctga cctgaaggag
gaggatggca gcctcagcct 2340ggatggtgca gacagcactg gcgtggtggc
caagctctca ccagccaacc agcggaaatg 2400tgagcgtgta ctgctggccc
tattctgtca cgaaccctgc cgccccctgc atcagctggc 2460taccgactcc
accttctccc tggaccagcc cggtggcacc ctggatctga ccctgatccg
2520tgcccgcctc caggagaagt tgtcacctcc ctacagctcc ccacaggagt
ttgcccagga 2580tgtgggccgc atgttcaagc aattcaacaa gttaactgag
gacaaggcag acgtgcagtc 2640catcatcggc ctgcagcgct tcttcgagac
gcgcatgaac gaggccttcg gtgacaccaa 2700gttctctgct gtgctggtgg
agcccccgcc gatgagcctg cctggtgctg gcctgagttc 2760ccaggagctg
tctggtggcc ctggtgatgg cccctgaggc tggagccccc atggccagcc
2820cagcctggct ctgttctctg tcctgtcacc ccatccccac tcccctggtg
gcctgactcc 2880cactccctgg tggccccatc ccccagttcc tcacgatatg
gtttttactt ctgtggattt 2940aataaaaact tcaccagtta aaaaaaaaaa
aaaaaaaaaa aaaaaaaaa 2989
* * * * *